Evaluation system for the efficacy of antimicrobial peptides and the use thereof

ABSTRACT

The present invention provides an evaluating system and the use thereof for the efficacy of antimicrobial peptide, which includes the following steps: (a) constructing a peptide by a first input unit, and load the peptide into an aqueous solution for a first time for equilibration; (b) constructing a lipid bilayer by a second input unit, and load the lipid bilayer into an aqueous solution for a second time for equilibration; (c) Using a first processing unit, simulations are carried out for an aqueous system containing an equilibrated peptide from the first input unit and the equilibrated lipid bilayer from the second input unit; (d) calculating the partition free energy of the peptide by a second processing unit; (e) outputting the prediction by an output unit, wherein the output unit is connected with the first processing unit and the second processing unit.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Non-provisional application claims priority under 35 U.S.C. §119(a) on Patent Application No(s). 104126710 filed in Taiwan, Republic of China Aug. 17, 2015, the entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention is related to an evaluating system for efficacy of antibiotic peptide and the use thereof, which is designed by stepwise circular reshuffling (SCR). By SCR, amino acids of known antibiotic peptides are collectively shifted in the primary sequence by one position at a time, in order to create a series of mutants. With the condition that the lengths of these mutants (or variants) are held unchanged, terminal residues can be shifted to the other end of the sequence depending on the direction of the shifting. The antibiotic activity and hemolytic activity of these peptide variants are evaluated by a computer simulation and a free energy calculation platform.

BACKGROUND OF THE INVENTION

Antibiotics can be classified into two groups. One contains secondary metabolites, produced by natural microorganisms (including bacteria, fungi, actinomyces) and able to inhibit the growth or survival of other microorganisms. The other contains chemically synthesized analogs of these metabolites. Antibiotic usually means anti-bacterial antibiotics in clinic but can also indicate antifungal and small pathogen antibiotics. Antibiotics now have been widely used and in fact abused causing a rampant spread of drug-resistant bacteria. Less and less long lasting antibiotics are there to battle against pathogen. Therefore, pharmaceutical companies have to constantly meet the pressing need to develop novel and more effective antibiotics.

According to the information from FierceBiotech, the global antibiotics market were expected to grow by a compound annual rate of 1.41% per year during 2013 to 2018. Although drug resistance problem caused some countries to partially ban antibiotics, the global market of antibiotic continues to expand for its irreplaceable medical benefit. According to the report of the Grand View Research, the global anti-infective agents market size was valued at USD 83.8 billion in 2015.

The antibiotic peptide, as known as antimicrobial peptide (AMP), the length of which is 10 to 80 amino acids and is what many organisms use to fight against pathogens through mechanisms of penetrating or perforating the bacterial cell membrane, interfering their proliferation and promoting wound healing. Therefore, AMPs have the potential to gradually replace antibiotics in the near future.

Most of the AMP developments involve replacing the amino acids on known antimicrobial peptides to possibly enhance their efficacy. It takes time to evaluate the effects of such replacements experimentally. Considering that antimicrobial activity results from complicated synergy of constituent residues, changing charge, hydrophobic moment, interaction energy and contacting surface area by mutagenesis in order to improve the efficacy may as well alter other needed antimicrobial functions such as secondary structures and amphiphilicity of AMPs. It is difficult to predict (even qualitatively) the antimicrobial activity from a single point mutation without conducting experiments. Here we provide a design method and computational evaluation platform to respectively create potent variants and assess variants' efficacy from molecular dynamics simulations and partition free energy calculations.

SUMMARY OF THE INVENTION

The present invention provides an antimicrobial, hemolytic and salt-resistant polypeptides that are designed by stepwise circular reshuffling (SCR). The approach involves (A) shifting constituent residues collectively along the primary sequence of peptides to create variants of different antimicrobial efficacy, strain specificity and hemolytic activity, and (B) a computational platform to evaluate the efficacy of these AMP variants using molecular dynamics simulations and partition free energy calculations.

The present invention provides a new platform based on molecular dynamics (MD) simulations and trajectory analysis to evaluate the antimicrobial and hemolytic activities of AMPs. This specific protocol involves a “sink and surface” simulation to pulled and forced the antimicrobial peptides to go into the biological membrane, which shortens the simulation time, as compared with the free diffusion method, by about 10 fold. The partition free energy, ΔG_(p), can be calculated from the simulation trajectory and was proven to be a better physical indicator to infer the efficacy of AMPs.

Accordingly, the present invention provides an evaluating system for the efficacy of antimicrobial peptide, which includes:

-   -   (a) a first input unit, wherein the first input unit is for         constructing a solvated peptide;     -   (b) a second input unit, wherein the second input unit is for         constructing a solvated lipid bilayer;     -   (c) a first processing unit, which is connecting with the first         input unit and the second input unit, is to perform molecular         dynamics (MD) simulations for the peptide in the first input         unit and the lipid bilayer in the second input unit;     -   (d) a second processing unit, connected with the first         processing unit, is to calculate <N_(i)> that is the moving         average number of heavy atoms of the antimicrobial peptide         contacting hydrophobic lipid tails and <N_(o)> that is the         moving average number of heavy atoms of the antimicrobial         peptide not contacting hydrophobic lipid tails during a 4 ns         window; and a partition free energy (ΔG_(p)) is calculated based         on <N_(i)> and <N_(o)>; wherein the heavy atoms are all the         non-hydrogen atoms;     -   (e) an output unit, connected with the second processing unit,         is used for outputting predicted information including the         partition free energy (ΔG_(p)),         -   wherein the first input unit, the second input unit, the             first processing unit, the second processing unit and the             output unit are operated by a computer.

A method for predicting antimicrobial efficacy of antimicrobial peptide by an in silico evaluating/prediction platform, comprises the following steps:

-   -   (a) A peptide is constructed by a first input unit, and is         equilibrated in an aqueous solution for a first time;     -   (b) A lipid bilayer is constructed by a second input unit, and         is equilibrated in an aqueous solution for a second time;     -   (c) a first processing unit, connecting with the first input         unit and the second input unit, performs molecular dynamics (MD)         simulations for the peptide in the first input unit and the         lipid bilayer in the second input unit;     -   (d) a second processing unit, connected with the first         processing unit, is to calculate <N_(i)> that is the moving         average of the number of heavy atoms in the antimicrobial         peptide contacting hydrophobic lipid tails and <N_(o)> that is         the moving average of the number of heavy atoms in the         antimicrobial peptide not contacting hydrophobic lipid tails         during a 4 ns window; and a partition free energy (ΔG_(p)) is         calculated based on <N_(i)> and <N_(o)>; wherein the heavy atoms         are all the non-hydrogen atoms;     -   (e) predicted partition free energy (ΔG_(p)) and/or predited         peptide activity are outputted by an output unit, wherein the         output unit is connected with the second processing unit.

Preferably, the conformation of the peptide is α-helix.

Preferably, the first input unit includes an equilibrated peptide initially taken from the X-ray crystallography or NMR data, or designed by a software including PyMol or Discovery Studio Visualizer; wherein the peptide is equilibrated by a simulation software that includes NAMD, VIVID, AMBER and GROMACS with commonly used force field that includes CHARMM, AMBER, GROMOS and OPLS under physiological conditions.

Preferably, the second input unit includes an“CHARMM-GUI” server to build the lipid bilayer; wherein the lipid bilayer is equilibrated by a simulation software that includes NAMD, VIVID, AMBER and GROMACS with commonly used force field including CHARMM, AMBER, GROMOS and OPLS under physiological conditions.

Preferably, the first processing unit uses a simulation software, which can be NAMD, VIVID, AMBER and GROMACS with commonly used force field including CHARMM, AMBER, GROMOS and OPLS to equilibrate the system where the peptides from the first input unit stably insert into the lipid bilayer from the second input unit.

Preferably, the input of the second processing unit (the number of heavy atoms in the peptide contacting with lipid tails inside the membrane) is obtained from the simulation trajectory outputted from the first processing unit.

Preferably, the lipid bilayer is composed by a palmitoyloleoyl phosphatidyl choline (POPC) and/or palmitoyloleoyl-phosphatidylglycerol (POPG).

Preferably, the first time duration is a few hundred picoseconds.

Preferably, the predicted information is the antimicrobial activity of the peptide.

Preferably, the molecular dynamics (MD) simulations is to first orient the hydrophobic side of the antimicrobial peptide to face a pre-equilibrated lipid bilayer; the “sink and surface” simulation is carried out after the projections of mass centers of the peptide and lipid bilayer on the lipid bilayer's normal being positioned 35 Å apart from each other.

Preferably, the “sink and surface” simulation is to pull the helical peptide by a weak force to where 6 Å below the average coordinate of the phosphor atoms in the upper leaflet of the lipid bilayer and to hold the peptide therein for several nanoseconds; afterwards, the weak restraint is released, and within 10 to 35 nanoseconds the helical peptide can float (surface) onto the membrane surface and equilibrate therein.

Preferably, the ΔGp=−k_(B)Tln(<Ni>/<No>) or =−k_(B)T(<lnNi>/<lnNo>), where k_(B) is the Boltzmann constant, T is the absolute temperature, Ni is the average number of heavy atoms of antimicrobial peptide contacting hydrophobic lipid tails and the No is the average number of heavy atoms of antimicrobial peptide that do not contact hydrophobic lipid tails; the < > symbol denotes moving averages for a 4 nanosecond window.

Preferably, in this invention, the correlation coefficient (R) of the partition free energy (ΔG_(p)) and corresponding bactericidal activity (in terms of minimal inhibitory concentration, or MIC) is found to be larger than 0.80 (“1” represents the perfect correlation). In this series, the MIC=13.49*ΔGp−5.79.

The present invention also provides a method to predict antimicrobial efficacy of antimicrobial peptide by the aforementioned evaluation platform, which comprises the following steps:

-   -   (a) A peptide is constructed by a first input unit, and the         peptide is loaded and equilibrated in an aqueous solution for a         first time;     -   (b) A lipid bilayer is constructed by a second input unit, and         the lipid bilayer is loaded and equilibrated in an aqueous         solution for a second time;     -   (c) a first processing unit, connecting with the first input         unit and the second input unit, performs molecular dynamics (MD)         simulations for the peptide in the first input unit and the         lipid bilayer in the second input unit;     -   (d) a second processing unit, connected with the first         processing unit, is to calculate <N_(i)> that is the moving         average of the number of heavy atoms in the antimicrobial         peptide contacting hydrophobic lipid tails and <N_(o)> that is         the moving average of the number of heavy atoms in the         antimicrobial peptide not contacting hydrophobic lipid tails         during a 4 ns window; and a partition free energy (ΔG_(p)) is         calculated based on <N_(i)> and <N_(o)>; wherein the heavy atoms         are all the non-hydrogen atoms;     -   (e) predicted partition free energy (ΔG_(p)) and/or predited         peptide activity are outputted by an output unit, wherein the         output unit is connected with the second processing unit.

Preferably, the antimicrobial peptide is designed according to the following formula: xAP1yBP2zCn(P3vD) (formula I); wherein the x, y, z, v and n are all positive integers; and x=0˜6, y=0 or 4, z=0˜6, v=0˜4 and n=0 or 1; wherein each of P1, P2 and P3 is -Trp-Leu-Lys-; wherein A, B and C are selected from the group of Trp, Leu and Lys, and D is Lys.

Preferably, the antimicrobial peptide is selected from the group of SEQ ID NO:2 to SEQ ID NO:13.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

The FIG. 1 is the simple figure of the present invention.

The FIG. 2 is the flow chart of the molecular dynamics (MD) simulations system of the present invention.

The FIG. 3 is the figure of the peptide insertion process observed in the molecular dynamics (MD) simulations system of the present invention.

The FIG. 4 is the figure of the hemolytic activities of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections that follow.

EXPERIMENTAL METHODS

The antimicrobial peptide of the present invention is as the following formula: xAP1yBP2zCn(P3vD) (formula I); where the x, y, z, v and n are all positive integers; and x=0˜6, y=0 or 4, z=0˜6, v=0˜4 and n=0 or 1; each of P1, P2 and P3 is -Trp-Leu-Lys-; A, B and C are selected from the group of Trp, Leu and Lys and D is Lys.

In the best embodiment, the result of molecular dynamics (MD) simulations for a series of AMP variants is shown in Table 1.

TABLE 1 the simulation results for an antimicrobial peptide family (SCR variants) Antimicrobial activity Simulation data (MIC) (μg/ml) Peptide ΔG_(P) Predicted Experimental numbers <Ni> <No> (kcal/mol) values values SEQ ID NO. 1 25.55 107.45 0.88 6.06 3.13 SEQ ID NO. 2 30.03 102.98 0.75 4.39 6.25 SEQ ID NO. 3 29.85 103.15 0.76 4.45 6.25 SEQ ID NO. 4 17.53 115.48 1.16 9.80 12.5 SEQ ID NO. 5 13.93 119.08 1.32 11.98 12.5 SEQ ID NO. 6 28.70 104.30 0.79 4.84 6.25 SEQ ID NO. 7 15.90 117.10 1.22 10.68 12.5 SEQ ID NO. 8 22.78 110.23 0.96 7.21 6.25 SEQ ID NO. 9 16.83 116.63 1.20 10.45 6.25 SEQ ID NO. 10 29.68 103.03 0.76 4.51 6.25 SEQ ID NO. 11 29.98 103.03 0.76 4.40 1.56 SEQ ID NO. 12 37.38 95.63 0.57 1.94 1.56 SEQ ID NO. 13 36.88 96.13 0.59 2.10 1.56 ΔG_(P) is the partition free energy.

In the best embodiment, the antimicrobial activity of the antimicrobial peptide is shown in Table 2.

MIC: Minimal Inhibitory Concentration of AMPs in MH Medium Against E. coli ATCC 25922

Minimal inhibitory concentration (MIC) represents for the lowest concentration of peptide at which the AMP can still kill 90% of the bacteria. All MIC tests for WLK peptide series SEQ ID NO:1-13 were measured against Escherichia coli strain (ATCC 25922) in triplicate. Peptide concentration was determined by UV spectrophotometer (Ultrospec 1100 pro from Amersham Biosciences) at 280 nm with proper extinction coefficient. (Gill S. C. & Von Hippel P. H., Analytical biochemistry 1989, 182(2), 319˜326). All of the samples were prepared from a stock solution containing peptides of concentrations 5, 2.5, 1.25, 0.625, 0.313, 0.156 and 0.078 mg/ml. Bacteria culture in the middle of logarithmic growth at a concentration of 5×10⁵ colony-formation unit (CFU) and protein solution at the aforementioned concentrations were uniformly mixed in a 96-well culture plate (each well contains 1 microliter protein solution and 99 microliter of bacteria culture). After growing the culture at 37° C. for 16 hours, how bacterial growth was inhibited was measured by Thermo Max (Molecular Devices) at the wavelength of 600 nm. The MIC values were determined by triplicates of the above experiments.

Hemolysis Activity Test

Hemolytic activity was determined by measuring hemolysis of human red blood cells. Fresh red blood cells and PBS buffer solution (pH 7.4) was mixed uniformly and centrifugated by relative centrigugal force (RCF) 800 g rpm for 10 minutes, watched and then diluted by the PBS buffer solution to 10% concentration by volume (v/v). All the tested peptides were prepared with different concentrations (800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56 μg/ml) and then mixed with PBS buffer in 1:1 ratio (100 μL:100 μL) by volume. In 1:1 ratio (v/v), the red blood cell solution was mixed with a PBS buffer, with or without 2% Triton X-100, respectively as the positive and negative controls. All samples to be tested were settled at 37° C. environment for 1 hour. Next, the specimen was centrifuged under RCF 800g for 10 minutes and the absorbance of its supernatant was measured at a wavelength of 450 nm. The hemolytic activity would be measured according to the following formula:

“% hemolysis=[A _(sample) −A _(PBS) ]/[A _(TritonX100) −A _(PBS)]”

TABLE 2 The hemolytic activity, salt resistance, and antimicrobial acitivity of the present invention MIC (μg/ml) MIC (μg/ml) in LYM medium in MH NaCl concentration (nM) AMPs Sequence <μH> medium control 50 100 200 300 SEQ ID NO: 1 KK WLK WLK WLK KK 0.647 3.13 1.56 1.56 3.13 6.25 6.25 SEQ ID NO: 2 K WLK WLK WLK KKK 0.506 6.25 1.56 1.56 6.25 12.5 12.5 SEQ ID NO: 3 WLK WLK WLK KKKK 0.575 6.25 1.56 3.13 3.13 12.5 25 SEQ ID NO: 4 LK WLK WLK KKKK W 0.417 12.5 1.56 3.13 3.13 12.5 50 SEQ ID NO: 5 K WLK WLK KKKK WL 0.663 12.5 3.13 3.13 12.5 25 50 SEQ ID NO: 6 WLK WLK KKKK WLK 0.700 6.25 1.56 3.13 6.25 12.5 25 SEQ ID NO: 7 LK WLK KKKK WLK W 0.454 12.5 3.13 3.13 6.25 12.5 50 SEQ ID NO: 8 K WLK KKKK WLK WL 0.682 6.25 3.13 3.13 3.13 6.25 12.5 SEQ ID NO: 9 WLK KKKK WLK WLK  0.678 6.25 3.13 3.13 3.13 12.5 50 SEQ ID NO: 10 LK KKKK WLK WLK W 0.371 6.25 3.13 3.13 3.13 6.25 25 SEQ ID NO: 11 K KKKK WLK WLK WL 0.555 1.56 1.56 1.56 1.56 1.56 3.13 SEQ ID NO: 12 KKKK WLK WLK WLK  0.518 1.56 1.56 1.56 3.13 3.13 3.13 SEQ ID NO: 13 KKK WLK WLK WLK K 0.661 1.56 1.56 3.13 1.56 3.13 12.5

The hemolytic activity, salt resistance, and antimicrobial activity of the novelly designed 12 isomeric peptides (SEQ ID NO: 2˜13) based on the original helical AMP of SEQ ID NO: 1 by the process of “stepwise circular reshuffling (SCR)” were shown in Table 2. The “sink and surface” simulation strategy and ΔGp calculation were used to measure the antimicrobial activity, and the relationship between (ΔGp) and antimicrobial activity (demonstrated by MIC) of the 13 isomeric peptides (including the original sequence) was analyzed, wherein the correlation coefficient was found greater than 0.8 (MIC=13.50*ΔGp−5.79, R=0.84). Table 2 shows that the 13 peptides in high salt concentration still had high inhibitory effects. Besides, 3 out of 12 peptides were found to have a better antimicrobial activity than the original sequence in the present invention, wherein SEQ ID NO: 12 showed lower hemolytic activity on human erythrocytes. As a result, the current invention provides a new platform whereby a repertoire of more effective antimicrobial peptides can be rationally designed based on known antimicrobial peptides, and their bactericidal activities can be predicted by herein presented computational methods. 

1. An evaluating system for the efficacy of antimicrobial peptides, includes: (a) a first input unit, wherein the first input unit is for constructing a peptide; (b) a second input unit, wherein the second input unit is for constructing a lipid bilayer; (c) a first processing unit, which is connecting with the first input unit and the second input unit, the first processing unit is conducting a molecular dynamics (MD) simulations in an aqueous solution with peptides constructed by the first input unit and the lipid bilayer constructed by the second input unit; (d) a second processing unit, connected with the first processing unit, is to calculate <N_(i)> that is the moving average of the number of heavy atoms in the antimicrobial peptide contacting hydrophobic lipid tails and <N_(o)> that is the moving average of the numbers of heavy atoms in the antimicrobial peptide not contacting hydrophobic lipid tails during a 4 ns window; and a partition free energy (ΔG_(p)) is calculated based on <N_(i)> and <N_(o)>; wherein the heavy atoms are all the non-hydrogen atoms; and (e) predicted partition free energy (ΔG_(p)) and/or predited peptide activity are outputted by an output unit, wherein the output unit is connected with the second processing unit; wherein the first input unit, the second input unit, the first processing unit, the second processing unit and the output unit are operated by a computer.
 2. The evaluating system of claim 1, wherein the first input unit includes a peptide that is designed by a software including PyMol or Discovery Studio Visualizer, or determined by experiments including X-ray crystallography or NMR; wherein the peptide is equilibrated in simulations conducted by simulation software including NAMD, VMD, AMBER, GROMACS with commonly used force field including CHARMM, AMBER, GROMOS and OPLS under physiological conditions.
 3. The evaluating system of claim 1, wherein the second input unit includes an “CHARMM-GUI” server to build the lipid bilayer; wherein the lipid bilayer is equilibrated in simulations conducted by simulation software including NAMD, VMD, AMBER, GROMACS with commonly used force field including CHARMM, AMBER, GROMOS and OPLS under physiological conditions.
 4. The evaluating system of claim 1, wherein the first processing unit that utilizes simulation software including NAMD, VMD, AMBER, GROMACS with commonly used force field including CHARMM, AMBER, GROMOS and OPLS to equilibrate inserted peptides in the lipid bilayer.
 5. The evaluating system of claim 1, wherein the lipid bilayer comprising palmitoyloleoyl phosphatidyl choline (POPC) and/or palmitoyloleoyl-phosphatidylglycerol (POPG).
 6. The evaluating system of claim 1, wherein the molecular dynamics (MD) simulation system that is to first make the hydrophobic side of the antimicrobial peptide face the pre-equilibrated lipid bilayer; then a sink and surface simulation is carried out after the projections of mass centers of the peptide and lipid bilayer on the normal of lipid bilayer being positioned 35 Å apart from each other.
 7. The evaluating system of claim 6, wherein the sink and surface simulate ion that is to pull the helical peptide by a weak force to where a few Ångstrom below the average coordinate of the phosphor atoms in the upper leaflet of the lipid bilayer and to hold the peptide therein for several nanoseconds; afterwards, the weak restraint is released, and within 10 to 35 nanoseconds the helical peptide can float (surface) onto the membrane surface and equilibrate therein.
 8. A prediction method for antimicrobial efficacy of antimicrobial peptides based on the evaluating system of claim 1, which comprises the following steps: (a) a first input unit, wherein the first input unit is for constructing a peptide; (b) a second input unit, wherein the second input unit is for constructing a lipid bilayer; (c) a first processing unit, which is connecting with the first input unit and the second input unit, the first processing unit is conducting a molecular dynamics (MD) simulations in an aqueous solution with peptides constructed by the first input unit and the lipid bilayer constructed by the second input unit; (d) a second processing unit, connected with the first processing unit, is to calculate <N_(i)> that is the moving average of the number of heavy atoms in the antimicrobial peptide contacting hydrophobic lipid tails and <N_(o)> that is the moving average of the numbers of heavy atoms in the antimicrobial peptide not contacting hydrophobic lipid tails during a 4 ns window; and partition free energy (ΔG_(p)) is calculated based on <N_(i)> and <N_(o)>; wherein the heavy atoms are all thenon-hydrogen atoms; and (e) predicted partition free energy (ΔG_(p)) and/or predited peptide activity are outputted by an output unit, wherein the output unit is connected with the second processing unit.
 9. The method of claim 8, wherein features a simulation where antimicrobial peptide is equilibrated in a solution for a few hundred picoseconds.
 10. The method of claim 8, wherein the first input unit includes a peptide that is designed by a software including PyMol or Discovery Studio Visualizer, or determined by experiments including X-ray crystallography or NMR; wherein the peptide equilibration is conducted by simulation software including NAMD, VMD, AMBER, GROMACS with commonly used force field including CHARMM, AMBER, GROMOS and OPLS under physiological conditions.
 11. (canceled)
 12. (canceled)
 13. (canceled)
 14. The prediction method of claim 8, further comprises predicting antimicrobial activity of the antimicrobial peptide.
 15. The prediction method of claim 8, wherein the molecular dynamics (MD) simulation system that is to first make the hydrophobic side of the antimicrobial peptide face the pre-equilibrated lipid bilayer, and a sink and surface simulation is then carried out after the projections of mass centers of the peptide and lipid bilayer on the normal of lipid bilayer being positioned 35 Å apart from each other.
 16. The prediction method of claim 8, wherein the sink and surface simulate ion that is to pull the helical peptide by a weak force to where a few Ångstrom below the average coordinate of the phosphor atoms in the upper leaflet of the lipid bilayer and to hold the peptide therein for several nanoseconds; afterwards, the weak restraint is released, and within 10 to 35 nanoseconds the helical peptide can float onto the membrane surface and equilibrate therein.
 17. The prediction method of claim 8, further comprising using partition free energy calculation of the quantity ΔGp=−k_(B)Tln(<Ni>/<No>) or =−k_(B)T(<lnNi>/<lnNo>), where k_(B) is the Boltzmann constant, T is the absolute temperature, Ni is the average number of heavy atoms of antimicrobial peptide contacting hydrophobic lipid tails and the No is the average number of heavy atoms of antimicrobial peptide that do not contact hydrophobic lipid tails; the < > symbol denotes moving averages of a 4 nanosecond window.
 18. An antimicrobial peptide with low hemolytic activities can be expressed by the following formula: xAP1yBP2zCn(P3vD)  (formula I); wherein the x, y, z, v and n are all positive integers; and x=0˜6, y=0 or 4, z=0˜6, v=0˜4 and n=0 or 1 each of P1, P2 and P3 is -Trp-Leu-Lys-; A, B and C are selected from the group of Trp, Leu and Lys and D is Lys.
 19. The antimicrobial peptide of claim 18, wherein the antimicrobial peptides are selected from a group of SEQ ID NO:2 to SEQ ID NO:13. 